Composition for treating and/or preventing Hepatitis B virus infection and the use thereof

ABSTRACT

A composition for treating and/or preventing Hepatitis B virus infection and Hepatitis B virus infection mediated diseases and the method thereof are provided. In some embodiments, the composition includes a polyriboinosinic acid-polyribocytidylic acid (PIC), at least one antibiotic or polyamide compound, at least one positive ion, and Hepatitis B virus surface antigen. In some embodiments, the composition includes PIC, at least one antibiotic or polyamide compound, at least one positive ion, Hepatitis B virus surface antigen and Hepatitis B virus core antigen. The present disclosure also relates to a method of treating and/or preventing Hepatitis B virus infection, particularly for treating chronic HBV infection.

RELATED APPLICATION

This application claims priority to Singapore application NO.10201706540X, filed on Aug. 10, 2017. The entire teachings of the aboveapplication are incorporated herein by reference.

TECHNICAL FIELD

This application relates to a composition for treating and/or preventingHepatitis B virus infection and Hepatitis B virus infection mediateddiseases. More particularly, this application relates to compositions,including a polyriboinosinic acid-polyribocytidylic acid (PIC), at leastone antibiotic or polyamide compound, at least one positive ion,Hepatitis B virus surface antigen, and/or Hepatitis B virus coreantigen. This application also relates to a method of treating and/orpreventing Hepatitis B virus infection or Hepatitis B virus infectionmediated diseases.

BACKGROUND

Hepatitis B virus (HBV) infection is an important global health problem.Nearly 2 billion of people are hepatitis B carriers, and about 400million of them are suffering from persistent infection (EuropeanAssociation for the Study of the Liver, EASL Clinical PracticeGuidelines: management of chronic hepatitis B virus infection. J.Hepatol. 2012. 57 (1):167-185). Liver cirrhosis, hepatic insufficiency,or liver cancer develop in 15%-40% of patients with chronic infection(Lok A S, McMahon B J, Chronic hepatitis B: update 2009. Hepatology.2009. 50 (3):661-662). It is estimated that 600,000 people die fromconsequences of the chronic infection every year, such as livercirrhosis and liver cancer (Lok A S et al., Chronic Hepatitis B.Hepatology. 2007. 45:507-539; World Health Organization, Hepatitis B(Fact sheet No. 204). 2013; National Institutes of H. NationalInstitutes of Health consensus development conference statement:Management of Hepatitis B. Ann Intern Med. 2009. 150:104-110).

Currently, 10 μg or 20 μg Hepatitis B virus surface antigen (HBsAg) isused in a preventive hepatitis B vaccine. However, 40 μg HBsAg may beadministered to patients who receive hemodialysis treatment orpre-transplantation patients (Mahoney F J., Update on Diagnosis,Management, and Prevention of Hepatitis B Virus Infection, Clin.Microbiol. Rev. 1999, 12:351-366; Bock M et al., Hepatitis B vaccinationin haemodialysis patients: A randomized clinical trial. Nephrology 2009;14:267-272; Somi M H et al., Improving hepatitis B vaccine efficacy inend-stage renal diseases patients and role of adjuvants. ISRNGastroenterol. 2012; 2012:960413; Bauer T and Jilg W., Hepatitis Bsurface antigen-specific T and B cell memory in individuals who had lostprotective antibodies after hepatitis B vaccination, Vaccine 2006;24:572-577). Under clinical conditions, it is safe to administer avaccine including 60 μg, 80 μs, or 90 μs HBsAg.

Hepatitis B virus core antigen (HBcAg), which is located on the internalcomponent of the HBV particle, is one indication of the HBV infection orHBV infection mediated diseases. United States patent application No.20170049817 disclosed preparation of a composition against HBV using amodified HBcAg. The entire content of the United States patentapplication No. 20170049817 is incorporated herein by reference.

Furthermore, some patients are susceptible to HBV, and show resistanceto standard immunization protocols. When receiving hemodialysistreatments, patients with end-stage renal diseases show higherprevalence rate and more adverse prognosis than normal people. However,after vaccination with HBV vaccines, patients with uremia orpre-transplantation patients show significantly lower seroconversionrate than that in individuals with immunity. Even though the protectiveantibodies are generated, peak antibody titers are low and duration ofimmune responses is also short (Bauer T. and Jilg W., Hepatitis Bsurface antigen-specific T and B cell memory in individuals who had lostprotective antibodies after Hepatitis B vaccination, Vaccine 2006;24:572-577; Bock M, Barros E and JV Veronese F, Hepatitis B vaccinationin haemodialysis patients: A randomized clinical trial, Nephrology 2009;14, 267-272).

When a vaccine including 40 μg HBsAg (double of generally used dosage)is administered to a dialysis patient, effect of the vaccine isunsatisfactory, and only about 50% to 60% of all vaccinated subjectshave developed protective antibodies (Mary S et al., High-Dose HepatitisB Vaccine in Patients Waiting for Lung Transplantation, Presentation atthe Fifth Annual Conference on Vaccine Research, Baltimore, Md., May 7,2002).

Furthermore, only 5% of adults have developed chronic infection afteracutely infected with HBV. If a subject is infected in perinatal period,the subject is more likely to get chronic infection. After first acuteinfection, 25%-30% of children below 5 years old have developed chronicinfection, and 90% of infants born by HBeAg positive mothers have riskfor acquiring chronic infection (Mahoney F J, Update on Diagnosis,Management, and Prevention of Hepatitis B Virus Infection, Clin.Microbiol. Rev. 1999; 12:351-366). Also, immunosuppressed patients afteran acute infection (for example, HIV infection) are prone to developchronic HBV infection (Mehta N et al., Impaired generation of hepatitisB virus-specific memory B cells in HIV infected individuals followingvaccination. Vaccine. 2010. 7; 28(21): 3672-8).

U. S. Food and Drug Administration (US FDA) has approved following drugsas alternatives of chronic HBV infection treatments: polyethyleneglycol(PEG) modified IFN-α, and nucleoside or nucleotide analogues (forexample, Lamivudine). Chronic HBV infection treatments and these drugsare used to arrest disease development, which means long-termadministration of antivirus drugs or PEG modified IFN-α is inevitable.

However, currently, an antivirus treatment often represents a failure.About 30% of the patients have achieved the goals of the treatment, andonly 10% of the patients are deemed to be cured. A long-term antivirustreatment is more likely to generate resistance to virus, and IFN-α canproduce significant side effects (Nebbia G et al., Hepatitis Binfection: current concepts and future challenges. QJM. 2012.105(2):109-13). Therefore, a treatment with adequate therapeutic effectsto chronic infection is needed.

Multi-specific antivirus CD4⁺ and CD8⁺ T cell responses are observed inpatients recovered from acute infection. These responses are strongerthan those in chronic patients, and virus-specific T cell responses inchronic patients are weak and instant (Webster, G. J et al.,Longitudinal analysis of CD8⁺ T cells specific for structural andnonstructural Hepatitis B virus proteins in patients with chronicHepatitis B: implications for immunotherapy. J Virol. 2004;78:5707-5719; Urbani, S et al., Acute phase HBV-specific T cellresponses associated with HBV persistence after HBV/HCV co-infection.Hepatology. 2005; 41:826-831).

Therefore, it is a potentially promising strategy to develop therapeuticvaccines by effectively activating CD4⁺ and CD8⁺ T cell immune responses(Sobao Y et al., J Hepatol 2002; 36:105-15; Sette A D et al., TheJournal of Immunology 2001; 166: 1389-1397; Chang, J. J et al., J Virol2005; 79:3038-3051; Guidotti L G et al., Science 1999; 284:825-9;Webster, G. J et al., Hepatology 2000; 32:1117-1124; Wieland, S. F. &Chisari, F. V. J Virol 2005; 79:9369-9380; Webster, G. J et al., J Virol2004; 78:5707-719; Urbani, S et al., Hepatology 2005; 41:826-31; Maini,M. K et al., J Exp Med 2000; 191:1269-1280).

It is reported that high-titer CD4⁺ and CD8⁺ T cell immune responses arecapable of removing virus in acute HBV infection (Mary S et al.,High-Dose Hepatitis B Vaccine in Patients Waiting for LungTransplantation, Presentation at the Fifth Annual Conference on VaccineResearch, Baltimore, Md., May 7, 2002; Rehermann B et al., NatureReviews Immunology 2005; 5:215-229; Bertoletti A et al., Journal ofHepatology 2003; 39:115-124; Bertoletti A et al., Journal of GeneralVirology 2006; 87:1439-1449; Guidotti L G et al., Ann Rev Immunol 2001;19:65-91; Ferrari C et al., J Immunol 1990; 145:3442-9; Penna A et al.,J Clin Invest 1996; 98:1185-94; Penna A et al., Hepatology 1997;25:1022-7; Rehermann B et al., J Exp Med 1995; 181:1047-58; Jung M etal., Virology 1999; 261:165-72).

According observations from preclinical studies, some methods arecapable of decreasing HBV DNA load. For example, one method isperforming short-term antivirus wash by Lamivudine before vaccination.For another example, some methods include eliciting T cell mediatedimmune responses to HBsAg vaccines (Bertoletti A et al., Curr OpinImmunol 2000; 12:403-8; Chisari F V. American Journal of Pathology 2000;156:1117-1132; Kakimi K et al., J Exp Med 2000; 192:921-30; Kakimi K etal., J Immunol 2001; 167:6701-5; Thimme R et al., J Virol 2003;77:68-76; Pol S et al., Lancet. 1994; 344:342; Mancini M et al., ProcNatl Acad Sci USA. 1996; 93:12496-12501).

Alum adjuvant is generally used to activate antibody mediated immunity,and helps build effective prevention against HBV infection. However alumadjuvant is not able to develop effective T cell mediated immuneresponses.

Polyriboinosinic acid-polyribocytidylic acid (PIC, also referring toPoly I:C) is proved to be a strong immunomodulator in animal tests, butnot in human tests, possibly due to fast degradation by human serumnucleases.

A stabilized PIC, also referring to PIKA (a composition including PIC,one or more antibiotics or polyamide compounds, one or more positiveions, and any other substance for preparing the PIKA), is capable ofbeing dissolved by solutions (PH: 6.0-8.0). PIKA is capable of enhancingproduction of neutralizing antibodies, and inducing T cell mediatedimmunity (Li L et al., Zhong Guo Mian Yi Xue Za Zhi 2006; 22:983-986).According to the present disclosure, PIC, antibiotics and positive ionsmay be those disclosed in WO2006/131023. The disclosure of WO2006/131023is incorporated herein by reference.

United States patent application publication NO. 20090136538A1 disclosesthat commercially available Hepatitis B vaccine is a liquid suspensionconsisting of purified HBsAg absorbed onto aluminum hydroxide adjuvant.Hepatitis B vaccine is a heat-stable vaccine, but a 50% loss of potencyof the vaccine has been reported to be observed after 9 months at 20° C.to 26° C., after 1 month at 36° C. to 40° C., and after 3 days at 45° C.So a stable Hepatitis B vaccine is needed to allow wider immunizationcoverage and to boost safety of the immunization, especially in somedeveloping countries or regions. The entire disclosure of United Statespatent publication NO. 20090136538A1 is incorporated herein byreference.

SUMMARY

A composition for treating and/or preventing Hepatitis B virus infectionor HBV infection mediated diseases, and a method of its use areprovided. In some embodiments, the composition includes PIC, at leastone antibiotic or polyamide compound, at least one positive ion, andHBsAg. In some embodiments, the composition includes PIC, at least oneantibiotic or polyamide compound, at least one positive ion, HBsAg andHBcAg.

In some embodiments, concentration of PIC in the composition ranges from250 μg/unit dose to 6000 μg/unit dose. In some embodiments, ratio ofHBsAg to PIC ranges from 1:50 to 1:5. In some embodiments, ratio of theHBcAg to PIC ranges from 1:50 to 1:5.

In some embodiments, the unit dose represented by volume ranges from 0.1ml to 250.0 ml.

According to one aspect of the present disclosure, the composition iscapable of eliciting protective antibody production, and/or CD4⁺ andCD8⁺ T cell mediated immune responses. The composition is capable ofpreventing HBV infection, alleviating HBV infection, arresting HBVinfection development, and/or removing HBV.

According to one aspect of the present disclosure, the composition iscapable of being administered to a HBV uninfected subject, a chronic HBVsubject, and an acute HBV subject, and eliciting or enhancing immuneresponses against HBV.

According to one aspect of the present disclosure, the composition isadministered to a subject at a frequency. In some embodiments, thefrequency may include once every two months, twice every two months,three times every two months, four times every two months, five timesevery two months, once every month, twice every month, once every threeweeks, twice every three weeks, twice every two weeks, once every twoweeks and once every week.

According to another aspect of the present disclosure, the presentdisclosure provides a kit. The kit includes at least one container. Insome embodiments, the container includes the composition, or at leastone component of the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is further described in terms of exemplaryembodiments. Some of these exemplary embodiments are described in detailwith reference to the drawings. These embodiments are non-limitingexemplary embodiments, in which like reference numerals representsimilar structures throughout the several views of the drawings, andwherein:

FIG. 1 illustrates production of anti-HBsAg IgG of mice afteradministration according to some embodiments of the present disclosure;

FIG. 2 illustrates production of anti-HBsAg IgG1 of mice afteradministration according to some embodiments of the present disclosure;

FIG. 3 illustrates production of anti-HBsAg IgG2a of mice afteradministration according to some embodiments of the present disclosure;

FIG. 4 is a diagram illustrating numbers of IFN-γ spot forming cells inspleen cells of mice after administration according to some embodimentsof the present disclosure;

FIG. 5 is a diagram illustrating numbers of IFN-γ spot forming cells inPBMC of health human subjects after administration according to someembodiments of the present disclosure;

FIG. 6 illustrates a flow cytometry spectrum of one subject infull-dosage group according to some embodiments of the presentdisclosure;

FIG. 7 illustrates PBMC cytokine production of vaccinated health humansubjects under stimulation of one single peptide according to someembodiments of the present disclosure;

FIG. 8 illustrates effects of the composition on HBV-transgene miceaccording to some embodiments of the present disclosure.

DETAILED DESCRIPTION

In the following detailed description, numerous specific details are setforth by way of examples in order to provide a thorough understanding ofthe relevant disclosure. However, it should be apparent to those skilledin the art that the present disclosure may be practiced without suchdetails. Various modifications to the disclosed embodiments are alsoapparent to those skilled in the art, and general principles definedherein may be applied to other embodiments and applications withoutdeparting from the spirit and scope of the present disclosure.

It will be understood that unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by those skilled in the art.

It will be understood that the singular forms “a”, “and”, and “the”include plural referents unless the context clearly dictates otherwise.As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items. It will be furtherunderstood that the terms “include”, and/or “comprise”, when used inthis disclosure, specify the presence of elements, steps, operations,and/or components, but not exclude the presence or addition of one ormore other elements, steps, operations, and/or components thereof.

It should be noted that unless indicated otherwise, term “treating”, or“treatment”, means alleviating HBV infection, arresting diseasedevelopment, and/or removing HBV by administering the composition andeliciting or enhancing immune responses. Unless indicated otherwise,term “preventing”, or “prevention”, means developing effective defenseagainst virus invasion by administering the composition and eliciting orenhancing immune responses.

The present disclosure provides a composition for treating and/orpreventing HBV infection. In some embodiments of the present disclosure,the composition includes PIC, at least one antibiotic or polyamidecompound, at least one positive ion, and HBsAg. In some embodiments ofthe present disclosure, the composition may further include HBcAg and/orany other component for implementing the composition.

It should be noted that components of the composition are not limiting.According to some embodiments of the present disclosure, the componentsof the composition are best defined by one or more, usually acombination of specific attributes. The specific attributes may includemolecular weight, molecular size, concentration, PH, dissolvability, orany other attributes in accordance with the present disclosure. Forexample, the composition may include several substances (e.g., sodiumbicarbonate) to adjust the PH of the composition.

According to some embodiments of the present disclosure, PIC moleculesof the composition are heterogeneous in molecular weight. The term“heterogeneous” used herein means the PIC molecules in the compositionhave different molecular weights. In some embodiments, the PIC moleculeswith their molecular weights equal to or greater than 66,000 Daltons(66,000 Daltons equals to 6.4 Svedbergs) may be used in the composition.For example, PIC molecules, of which molecular weights are between66,000 Daltons and 1,200,000 Daltons (i.e., between 6.4 and 24.0Svedbergs), may be used in the composition. For another example, themolecular weights of the PIC molecules may be greater than 150,000Daltons. In some embodiments, molecular weights of the PIC molecules mayrange from 100,000 Daltons to 200,000 Daltons, from 300,000 Daltons to4,000,000 Daltons, from 500,000 Daltons to 1,000,000 Daltons, from1,000,000 Daltons to 1,500,000 Daltons, from 1,500,000 Daltons to2,000,000 Daltons, from 2,000,000 Daltons to 2,500,000 Daltons, from2,500,000 Daltons to 3,000,000 Daltons, from 3,000,000 Daltons to3,500,000 Daltons, from 3,500,000 Daltons to 4,000,000 Daltons, from4,000,000 Daltons to 4,500,000 Daltons, or from 4,500,000 Daltons to5,000,000 Daltons.

According to some embodiments of the present disclosure, the antibioticin the composition may be tobramycin, anthracycline, butyrosin sulfate,gentamicin, hygromycin, amikacin, dibekacin, nebramycin, beta-lactam,neomycin, puromycin, streptomycin, streptozotocin, kanamycin, or thelike, or any combination thereof. In some embodiments, the antibioticmay be kanamycin.

According to the present disclosure, concentration of the antibiotic mayrange from 10 unit/ml to 100,000 unit/ml. In one particular embodiment,the concentration of the antibiotic may range from 100 unit/ml to 10,000unit/ml. In another particular embodiment, the concentration of theantibiotic may range from 500 unit/ml to 5,000 unit/ml. It should benoted that the concentration may be set according to several attributes(e.g., side-effects of long-term or high-dosage antibiotic use,induction of anti-antibiotic microorganism, etc.). In some embodiments,the concentration of the antibiotic (e.g., kanamycin) is not greaterthan 1000 IU/ml according to preclinical safety and toxicologyevaluation.

It should be noted that to those skilled in the art, the antibiotic andthe concentration are not limiting. In some embodiments, the antibioticand the concentration may be set according to characteristics of thesubject (e.g., age, antibiotic allergy, etc.) and any othercharacteristics. For example, for children under 5 years old, theconcentration of the antibiotics may decrease. For example, for subjectsallergic to ampicillin-like antibiotics, the antibiotic in thecomposition may be non-ampicillin antibiotic (e.g., kanamycin, etc.)

According to some embodiments of the present disclosure, the positiveion is provided by a salt or complex, including an organic or inorganicsalt or complex (e.g., chloride, fluoride, hydroxide, phosphate,sulfate, etc.). For example, the positive ion may be calcium, which maybe provided by calcium carbonate, calcium chloride, calcium fluoride,calcium hydroxide, calcium phosphate, or calcium sulfate. Exemplarypositive ions may include, but not limited to, calcium, cadmium,lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium,gallium, iodine, iron, and/or zinc. In one particular embodiment, thepositive ion may be calcium. According to some embodiments of thepresent disclosure, concentration of the positive ion may range from0.01 μmol/unit dose to 10 mmol/unit dose. In one particular embodiment,the concentration of the positive ion ranges from 0.02 μmol/unit dose to5 mmol/unit dose. In another particular embodiment, the concentration ofthe positive ion ranges from 0.1 μmol/unit dose to 1 mmol/unit dose. Inone more particular embodiments, the concentration of the positive ionranges from 0.1 μmol/unit dose to 100 μmol/unit dose.

According to some embodiments of the present disclosure, concentrationof the PIC may range from 250 μg/unit dose to 6000 μg/unit dose. In someembodiments, the concentration of the PIC may be 250 μg/unit dose, 500μg/unit dose, 1000 μg/unit dose, 1500 μg/unit dose, 2000 μg/unit dose,3000 μg/unit dose, 4000 μg/unit dose, 5000 μg/unit dose, or 6000 μg/unitdose. The concentration of the PIC is not limiting. The concentration ofthe PIC may be any value between 250 μg/unit dose and 6000 μg/unit dose.For example, the concentration of the PIC may range from 500 μg/unitdose to 4000 μg/unit dose, 1000 μg/unit dose to 3000 μg/unit dose, 1000μg/unit dose to 2500 μg/unit dose.

According to some embodiments of the present disclosure, when thecomposition is administered to adults, the concentration of the PIC maybe 500 μg/unit dose, 1000 μg/unit dose, 1500 μg/unit dose, 2000 μg/unitdose, or any value between 500 μg/unit dose and 2000 μg/unit dose. Whenthe composition is administered to juveniles (e.g., children), theconcentration of the PIC may be 250 μg/unit dose, 500 μg/unit dose, 1000μg/unit dose, 1250 μg/unit dose, or any value between 250 μg/unit doseand 1250 μg/unit dose.

According to some embodiments of the present disclosure, the unit dosemay represent weight, volume, or any other characteristic of aparticular dose of the composition. For example, the unit dose mayrepresent volume of one individual package (e.g., a tablet) of thecomposition. The unit dose may represent weight, volume, or any othercharacteristic of a component of the composition. For example, the unitdose may represent concentration of the PIC or PIKA in one particulardose of the composition. In some embodiments, the unit dose mayrepresent volume of one particular dose of the composition. Merely byway of example, value of the unit dose represented by volume of oneindividual package may be 0.1 ml, 0.15 ml, 0.2 ml, 0.5 ml, 1.0 ml, 1.5ml, 2.0 ml, 2.5 ml, 3.0 ml, 4.0 ml, 5.0 ml, 10.0 ml, 20.0 ml, 30.0 ml,40.0 ml, 50.0 ml, 60.0 ml, 70.0 ml, 80.0 ml, 90.0 ml, 100.0 ml, 150.0ml, 200.0 ml, 250.0 ml, or any value between 0.1 ml and 250.0 ml. It isunderstood to those skilled in the art, high or low unit dose may not beconvenient for clinical operations. The value of the unit dose may beset according to some attributes including administration methods (e.g.,nasal delivery, intravenous injection, oral delivery, etc.),characteristics of subjects (e.g., human, body weight, vaccinationhistory, etc.), characteristics of the composition (e.g., concentrationof the PIC, PH of the composition, etc.), and/or any other attribute.For example, when the composition is administered by injection to humansubjects or other subjects with similar height or similar weight, valueof the unit dose may range from 0.5 ml to 1.0 ml. For another example,when the administration method is nasal delivery, value of the unit dosemay range from 0.15 ml to 0.2 ml. More particularly, for anotherexample, when the composition is administered by intravenous injectionto human subjects or other subjects with similar size or similar weight,value of the unit dose may range from 30.0 ml to 100.0 ml. It should benoted that although the unit dose used herein represents volume of thecomposition, it does not mean the composition is limited to a liquidcomposition. For example, when the composition is in the solid form(e.g., dry powder, freeze-dried powder, etc.) and may be furtherprepared into a liquid form, the unit dose may represent volume of thecomposition in the liquid form.

According to some embodiments of the present disclosure, the compositionmay further include substances to stabilize the composition. Merely byway of example, the substances may include gelatin, saccharose,granulated sugar, lactose, maltose, trehalose, glucose, low moleculardextran, sorbitol, polysorbate 20, mannitol PEG, human blood albumin,recombinant human serum albumin, sodium caprylate, urea, aluminumhydroxide, phenol red, magnesium chloride, potassium chloride, sodiumchloride, sodium thiosulphate, potassium dihydrogen, ascorbic acid,trichloromethane, phenol, thiomersal, or the like, or any combinationthereof.

According to some embodiments of the present disclosure, the compositionmay further include a physiologically acceptable buffer. Merely by wayof example, the physiologically acceptable buffer may include acetatebuffer, trisamine buffer, bicarbonate buffer, carbonate buffer,phosphate buffered saline, or the like, or any combination thereof. PHvalue of the physiologically acceptable buffer may range from 6.0 to8.0. In some embodiments, PH value of the physiologically acceptablebuffer may be 6.0, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or any value between 6.0 and 8.0. In someparticular embodiments, the physiologically acceptable buffer isphosphate buffered saline (PH: 7.3-7.5).

According to some embodiments of the present disclosure, the compositionmay be in the form of dry powder, solution (e.g., parenteral solution,aqueous solution, saline solution, suspension, ointment, drop, emulsion,gel, syrup, serous, etc.), tablet, coated tablet, suppository, pill,granule, sugar-coated tablet, capsule, or the like, or any combinationthereof. In some embodiments, the composition may be prepared into aparenteral solution. The form of the composition is not limiting. Formsof a composition and their preparation described in the related art(e.g., Stanley A Plotkin et al., Vaccine, 4^(th) edition, W.B. SaundersCompany 2003) are within the spirit and scope of the present disclosure.

According to some embodiments of the present disclosure, HBsAg may beproduced by organisms including Hansenula Polymorpha, Chinese hamsterovary cell (CHO cell), Pichia pastoris, insect expression system, or thelike, or any combination thereof. The HBsAg with purity greater than 99%may be administered to infants, children over 5 years old, and adults.According to some embodiments of the present disclosure, the HBcAg maybe produced by recombinant DNA methods. The HBcAg may be produced byEscherichia coli, Hansenula Polymorpha, Pichia pastoris, Saccharomycescerevisiae, insect expression system, or the like, or any combinationthereof.

In some embodiments, weight ratio of HBsAg to the PIC may be 1:50, 1:40,1:30, 1:25, 1:20, 3:50, 1:15, 1:10, 1:5, or any value between 1:50 and1:5. In some particular embodiments, the weight ration of HBsAg to thePIC may be 1:25. It should be noted that the weight ratio of HBsAg tothe PIC is not limiting. For example, a composition with weight ratio ofHBsAg to the PIC near 1:25 (e.g., 10% lower or higher) is capable ofproviding adequate safety, and eliciting earlier also stronger (i.e.,antibodies of higher titer are generated) humoral immune responses and Tcell mediated immune responses.

In some embodiments, concentration of the HBsAg in the composition mayrange from 10 μg/unit dose to 100 μg/unit dose. Merely by way ofexample, the concentration of the HBsAg in the composition may be 10μg/unit dose, 15 μg/unit dose, 20 μg/unit dose, 25 μg/unit dose, 30μg/unit dose, 35 μg/unit dose, 40 μg/unit dose, 50 μg/unit dose, 60μg/unit dose, 70 μg/unit dose, 80 μg/unit dose, 90 μg/unit dose, 100μg/unit dose, or any value between 10 μg/unit dose and 100 μg/unit dose.In some embodiments, the concentration of the HBsAg in the compositionmay be 20 μg/unit dose, 40 μg/unit dose, or 60 μg/unit dose.

In some embodiments, concentration of the HBcAg in the composition mayrange from 10 μg/unit dose to 100 μg/unit dose. Merely by way ofexample, the concentration of the HBcAg in the composition may be 10μg/unit, 15 μg/unit dose, 20 μg/unit dose, 25 μg/unit dose, 30 μg/unitdose, 35 μg/unit dose, 40 μg/unit dose, 50 μg/unit dose, 60 μg/unitdose, 70 μg/unit dose, 80 μg/unit dose, 90 μg/unit dose, 100 μg/unitdose, or any value between 10 μg/unit dose and 100 μg/unit dose. In someembodiments, the concentration of the HBcAg may range from 10 μg/unitdose to 50 μg/unit dose, or from 50 μg/unit dose to 100 μg/unit dose.

According to some embodiments of the present disclosure, the compositionmay be administered to HBV uninfected subjects, subjects with chronicHBV infection (also referring to “chronic subjects” or “chronic HBVsubjects” in the present disclosure), and subjects with acute HBVinfection (also referring to “acute subjects” or “acute HBV subjects”).In some embodiments, the composition of the present disclosure is ableof eliciting stronger CD4⁺ T cell and CD8⁺ T cell immune responses thana combination of antigen and alum adjuvant. For example, the compositionof the present disclosure may elicit higher cytokine production (2-30times) than the combination of antigen and alum adjuvant.

In one particular embodiment, the composition may include 20 μg/unitdose HBsAg, 20 μg/unit dose HBcAg and 500 μg/unit dose PIC. In anotherparticular embodiment, the composition may include 40 μg/unit doseHBsAg, 40 μg/unit dose HBcAg and 1000 μg/unit dose PIC.

According to one aspect of the present disclosure, the composition maybe used for treating and/or preventing HBV infection, preferably chronicHBV infection.

According to one aspect of the present disclosure, the composition fortreating and/or preventing HBV infection is in the form of dry powder orfreeze-dried powder.

According to one aspect of the present disclosure, the composition maybe used to manufacture medicines for treating and/or preventing HBVinfection. In some embodiments, the medicines may include preventivevaccines, therapeutic vaccines, vaccine preparations, or the like, orany combination thereof. In one particular embodiment, the compositionmay be used to prepare medicines for treating and/or preventing chronicHBV infection.

According to another aspect of the present disclosure, a method fortreating and/or preventing HBV infection is provided. The method may beconfigured according to characteristics of the composition (e.g., unitdose, PH, etc.), administration methods (e.g., oral delivery, nasaldelivery, intravenous injection, etc.), characteristics of subjects(e.g., age, gender, antibiotic allergy, etc.), purpose of use (e.g.,treatment or prevention against HBV infection), and/or any otherattribute for implementing administration of the composition to thesubjects. Merely by way of example, in some embodiments, when thecomposition is used to treat HBV infection, the method may includeadministering the composition of a treatment effective amount to thesubjects. Similarly, when the composition is used to prevent HBVinfection, the method may include administering the composition of aprevention effective amount to the subjects. The term “treatmenteffective amount” used herein refers to an amount enough to elicit Tcell mediated immune responses and alleviate or remove infection. Theterm “prevention effective amount” used herein refers to an amountenough to elicit protective antibodies against virus infection. Forexample, as recommended by World Health Organization (WHO),concentration of the protective antibodies may be at least 10 mIU/ml.

In some embodiments, the method may be configured according tocharacteristics of the composition. The characteristics of thecomposition may include unit dose, PH of the composition, or the like.For example, the method may include adjusting the PH of the compositionto 6.0-8.0 by adding a physiological acceptable buffer into thecomposition. For another example, the method may include administratingthe composition of a certain amount (e.g., an amount smaller, greater,or equal to a unit dose) to subjects according to value of the unitdose.

In some embodiments, the method may be configured according toadministration methods. For example, the administration may besystematically or locally. The administration methods may includeparenteral injection (e.g., intramuscular injection, intraperitonealinjection, intravenous injection, subcutaneous injection, intradermaldelivery, intratumor delivery, peritumoral delivery, etc.), transdermaldelivery, and any other way of administration for implementing themethod. More particularly, in some embodiments, the administrationmethods may further include rectal delivery, vagina delivery, nasaldelivery, oral delivery, opthamalical delivery, sublingual delivery, andinhalation. It should be noted that the administration methods are notlimiting. To those skilled in the art, various modifications and changesare still within the sprite and scope of the present disclosure.

In some embodiments, the method may be configured according to frequencyof administration. The frequency of administration may be once every twomonths, two times every two months, three times every two months, fourtimes every two months, five times every two months, once every month,twice every month, once every three weeks, twice every three weeks,twice every two weeks, once every two weeks, once every week, or anyother suitable frequency for implementing the method. For example, whenthe composition is administered to HBV uninfected subjects, acomposition of a prevention effective amount may be administered to thesubjects three times every two months, twice every month, or three timesevery six months. For another example, when the composition isadministered to HBV infected subjects, a composition of a treatmenteffective amount may be administered to the subjects four times everytwo months, five times every two months, two times every month, onceevery three weeks, two times every three weeks, or once every week.According to the present disclosure, a month equals to 28, 29, 30, or 31days, and one week equals to 7 days.

In some particular embodiments, the method may include administrating(e.g., by intramuscular injection) the composition to a subject threetimes every two months. For example, the method may include followingsteps:

1) on day 0, administering the composition of a first amount to asubject;

2) from day 25 to day 31, administering the composition of a secondamount to the subject;

3) from day 53 to day 59, administering the composition of a thirdamount to the subject.

The first amount, the second amount, and the third amount may be thesame, or different with each other. For example, at least one of thefirst amount, the second amount and the third amount equals to atreatment effective amount, or a prevention effective amount. Foranother example, at least one of the first amount, the second amount andthe third amount is set according to concentration of HBsAg (e.g., 20μg/unit dose, 40 μg/unit dose, etc.), concentration of HBcAg (e.g., 10μg/unit dose, 20 μg/unit dose, 50 μg/unit dose, 100 μg/unit dose, etc.),and/or concentration of PIC (e.g., 500 μg/unit dose, 1000 μg/unit dose).In some embodiments, at least one of the first amount, the second amountand the third amount includes 20 μg/unit dose HBsAg and 500 μg/unit dosePIC. In some embodiments, at least one of the first amount, the secondamount, and the third amount includes 40 μg/unit dose HBsAg and 1000μg/unit dose PIC. In some embodiments, at least one of the first amount,the second amount, and the third amount includes 10 μg/unit dose to 50μg/unit dose HBcAg. In some embodiments, at least one of the firstamount, the second amount, and the third amount includes 50 μg/unit doseto 100 μg/unit dose HBcAg.

According to some embodiments of the present disclosure, a kit fortreating and/or preventing HBV infection is provided. The kit mayinclude at least one of one container, one needle, water for injection,a manual, or the like, or any combination thereof. For example, the kitmay include three containers. The composition contained in thecontainers may include the same or different formation and/or amount ofthe composition. For example, at least one container (e.g., two, orthree containers) may include 20 μg/unit dose HBsAg and 500 μg/unit dosePIC. At least one container (e.g., two, or three containers) may include10 μg/unit dose to 20 μg/unit dose HBcAg. For another example, at leastone container (e.g., two, or three containers) may include 40 μg/unitdose HBsAg and 1000 μg/unit dose PIC. At least one container (e.g., two,or three containers) may include 50 μg/unit dose to 100 μg/unit doseHBcAg. In some other embodiments, the composition in one container maybe in a liquid form (e.g., suspension, solution, etc.). In someembodiments, the composition in one container may be in a solid form(e.g., dry powder, freeze-dried powder, etc.). The composition in solidform may be prepared into a liquid form (e.g., suspension, solution,etc.), and stored in one or more sterilized containers (e.g., a tube, abottle, an ampoule, a syringe, etc.) before use.

EXAMPLES Example 1: Preparation of the Compositions

In some embodiments, the composition includes HBsAg, PIC, kanamycin, andcalcium chloride, where ration of the HBsAg to PIC is 1:25. Moreparticularly, the composition includes 40 μg/unit dose HBsAg, 1000μg/unit dose PIC, 800 IU/unit dose kanamycin, and 0.16 μmol/unit doseCa²⁺.

In some embodiments, the composition includes HBcAg, HBsAg, PIC,kanamycin, and calcium chloride. The composition includes 40 μg/unitdose HBsAg, 1000 μg/unit dose PIC, 800 IU/unit dose kanamycin, and 0.16μmol/unit dose Ca²⁺. More particularly, concentration of the HBcAgranges from 10 μg/unit dose to 50 μg/unit dose.

In some embodiments, the composition includes HBcAg, HBsAg, PIC,kanamycin, and calcium chloride. The composition includes 40 μg/unitdose HBsAg, 1000 μg/unit dose PIC, 800 IU/unit dose kanamycin, and 0.16μmol/unit dose Ca²⁺. More particularly, concentration of the HBcAgranges from 50 μg/unit dose to 100 μg/unit dose.

Example 2: Preclinical Toxicology Studies

EXAMPLE 2 illustrates preclinical toxicology studies of the compositionand its main components (including PIC, at least one antibiotic orpolyamide compound, and at least one positive ion, collectively, “PIKA”)according to some embodiments of the present disclosure. As illustratedin Table 1, mice used in EXAMPLE 2 have an average body weight of 0.019kg, and can tolerate 0.2 ml of the composition which includes 8 μg HBsAg(0.4 mg/kg) and 200 μg PIC (10 mg/kg), or tolerate 0.2 ml of the PIKAwhich includes 200 μg PIC (10 mg/kg). Dosage of the composition used inthe mice is about 1,200 times higher than that recommend dosage used ina human being (20 μg HBsAg (0.0003 mg/kg) and 500 μg PIKA (0.0083mg/kg)). Recommended maximum dosage used in a human being is double ofthe recommended minimum dosage, which provides a Margin of Safety (MoS)of 600. According to chronic toxicology studies of rodent rats, valuesof the MoS are 600 to recommended minimum dosage in human, and 300 torecommended maximum dosage in human, respectively. According topreclinical studies of primates, values of the MoS are 40 to recommendedminimum dosage in human, and 20 to recommended maximum dosage in human,respectively.

TABLE 1 MoS in preclinical toxicology studies MoS of PIC MoS of theSafety or PIKA composition Acute toxicology of rodents Recommendedminimum dosage in human 1,200 1,200 Recommended maximum dosage in human600 600 Chronic toxicology of rodents Recommended minimum dosage inhuman 600 600 Recommended maximum dosage in human 300 300 Chronictoxicology of primates Recommended minimum dosage in human 40 40Recommended maximum dosage in human 20 20 Considering body weight of anexemplary human subject is 60 kg, MoS is defined as follows: MoS =(dosage used in preclinical studies, mg/kg) ÷ (recommended dosage inhuman, mg/kg)

Example 3: Preclinical Studies of Immune Responses of Rodents

Methodology

Rodents used in EXAMPLE 3 are BALB/c mice of 6 weeks old, which aredivided into 6 groups. Each group includes 18 mice (9 males, with bodyweight ranging from 17.3 g to 20.7 g; 9 females, with body weightranging from 19.0 g to 23.7 g). As illustrated in Table 2, thecomposition and other substances are administered to the mice on day 1,day 8, day 22, day 36, day 50, day 64 and day 78 by tibialis anteriorinjection.

TABLE 2 Administration schedule adjuvant control BALB/c HBsAg PIKA AlumPBS Group Group name mice (μg) (μg) (μg) (μl) 1 Solvent control 9M, 9F —— — 250 group 2 Antigen control 9M, 9F 15 — — — group 3 Vaccine control9M, 9F 3 — 80 — group 4 Low-dosage 9M, 9F 3 50 — — group 5 Medium-dosage9M, 9F 9 150 — — group 6 High-dosage 9M, 9F 15 250 — — group

On day 3, day 24, day 38, day 52, day 66, day 80, and day 106, bloodsamples are taken for measurement of titer of anti-HBsAg antibodies, inorder to determine humoral immune responses.

On day 38, day 80, and day 106, three males and three females from eachgroup are sacrificed in order to determine T cell mediated immuneresponses. HBsAg or HBsAg CD8⁺ peptide is used for in vitro stimulationof spleen cells. Frequency of the spleen cells producing IFN-γ ismeasured by ELISPOT.

Results

As illustrated in FIG. 1 , FIG. 2 , and FIG. 3 , the composition(including HBsAg and PIKA) elicits stronger humoral immune responses(represented by production of IgG, IgG1, and IgG2a), which indicatesPIKA significantly enhances immune responses against HBsAg.

According to FIG. 3 , the composition produces strong Th1 type immuneresponse and significantly increases production of IgG2a.

According to FIG. 4 , results of the ELISPOT experiments indicate thatnumbers of the spleen cells producing IFN-γ significantly increase afterthe in vitro stimulation. The composition (including HBsAg and PIKA)significantly enhances T cell mediated immune responses.

Increasing value of IgG2a titer indicates that main immune responseselicited by the composition belong to Th1 type immune responses. Th1type immune responses and cellular immune responses are importanteffects of therapeutic vaccines. The Th1 type immune responses preventvirus infection, and remove infected cells. In addition, increasingnumber of the spleen cells producing IFN-γ indicates that thecomposition significantly enhances cellular immune responses.

Example 4: Chronical Toxicology Studies of Primates

Laboratory:

EXAMPLE 4 is conducted at National Center for Safety Evaluation ofDrugs, National Institute for the Control of Pharmaceutical andBiological Products, P.R. China.

Methodology

The primates used in EXAMPLE 4 are Rehsus monkeys divided into fivegroups. Each group includes 8 Rehsus monkeys (4 females and 4 males;1.6-3.2 years old; 3-3.5 kg). The composition, HBsAg, PIKA or PBS isadministered to the primates 5 times (on day 0, day 14, day 28, day 56,and day 84) in three months according to Table 3.

TABLE 3 Grouping and administration schedule HBsAg PIKA Concen- Concen-Dosage tration Dosage tation Group Animal (μg) (mg/kg) (μg) (mg/kg)Control 1 4M, 4F — — 1,000 0.31 — 2 4M, 4F 40 0.012 — — — 3 4M, 4F 400.012 1,000 0.31 — 4 4M, 4F 60 0.018 1,500 0.46 — 5 4M, 4F — — — — PBS

Each group receives 40 μg HBsAg and 1,000 μg PIC (or PIKA), or 60 μgantigen HBsAg and 1,500 μg PIC (or PIKA).

Local (e.g., administration site) and systematic clinical symptoms areobserved, and blood samples are taken 5 times from day 2 to day 126. Onday 85 (i.e., 24 hours after fifth administration) and day 126, themonkeys are sacrificed. 36 organs of the female monkeys and 32 organs ofthe male monkeys are used for histological examinations.

Histological Examinations

The histological examinations include:

-   -   clinical symptoms,    -   appearance, hair, activity, reaction, respiration, posture,        head-shoulder (including eyes, ears, mouth, nose), abdomen,        anus, perineum, skin color, muscle tension, trauma, tumor;    -   symptom related behaviors;    -   symptoms of administration site;    -   body weight;    -   feeding;    -   body temperature;    -   hematology: T, APTT, RBC, WBC, HGB, HCT, MCV, MCH, MCHC, PLT,        MPV, reticulocytes count %, Neut %, Lymph %, Mono %, Eos %, Baso        %;    -   biochemical tests: AST, ALT, BUN, CHO, GLU, TBIL, CRE, ALP, CK,        LDH, TP, ALB, TG, GGT, Ca, P, K⁺, Na⁺, Cl⁻, IgA, IgG, IgM, C3,        C4;    -   urine test;    -   pathological and histological analysis of the following organs:        brain, spine cord, heart, artery, lung (including bronchus),        liver, kidneys, spleen, pancreas, stomach, duodenum, jejunum,        ileum, colon, rectum, caecum, testis, epididymis, prostate,        ovary, uterus, vagina, bladder, pituitary gland, thyroid,        parathyroid, submandibular gland, adrenal gland, sciatic nerve,        muscles, mesenteric lymph nodes, inguinal lymph nodes, thymus,        mammary gland, sternum, administration site;    -   humoral immune response: antibody titers in serum measured by        ELISA.

Results

No local or systematic symptoms are observed. No abnormal changes areobserved in hematologic tests and serum biochemical tests. Results ofspleen histological test show increased numbers of activated macrophagesand mitosis in germinal centers of spleen white pulp. The germinalcenters of spleen white pulp are main areas of lymphocyte proliferation,especially main areas of B cell activation after antigen stimulation.PIKA enhances immune responses against antigen, and elicits lymphocyteproliferation. No spleen necrosis or cell death is observed. Therefore,the results indicate histological changes of spleen are notpathological, but physiological; the immune responses are reversible.Abnormal phenomena are not observed in histological results of any otherorgans.

The antibody titers in serum indicate the primates develop enhancedhumoral immune responses after administration of a composition includingHBsAg and PIKA. Table 4 illustrates immune response of the primates.

TABLE 4 Antibody titers in serum PIC or HBsAg PIKA Antibody titers Group(mg/kg) (mg/kg) Day 0 Day 21 Day 43 Day 85 Day 100 Day 126 1 — 0.31 0 00 0 0 0 2 0.012 — 0 2 6 148 620 253 3 0.012 0.31 0 72 444 1585 4502 23794 0.018 0.46 0 140 602 5261 11459 4912 5 PBS 0 0 0 0 0 0

Example 5: Clinical Studies of the Composition in Health Human Subjects

Laboratory

Department of Gastroenterology and Hepatology of Singapore GeneralHospital, Singapore; Clinical Trials & Research Unit of Changi GeneralHospital, Singapore.

Methodology

Health human subjects are randomly divided into three groups. Two ofthree groups receive increasing dose of the composition, and the thirdgroup receives a commercially available HBV vaccine. The composition andvaccine are administered as an intramuscular injection in the deltoidregion of the upper arm.

Composition and Vaccine

Group A (or “half dosage” group): the composition including 20 μg HBsAgand 500 μg PIKA;

Group B (or “full dosage” group): the composition including 40 μg HBsAgand 1000 μg PIKA;

Group C (or “control” group): a vaccine including 20 μg HBsAg and 500Alum adjuvant (e.g., commercially available vaccine from GSK:ENGERIX™-B)

Administration Schedule

To implement double-blind tests, the subjects receive the composition orthe vaccine three times and placebo once in 6 months as illustrated inTable 5.

TABLE 5 Administration schedule Day Day Day Day Day Day Day group 0 7 2856 84 168 196 A&B x x x ∘ C x x ∘ x x means administration of thecomposition or the vaccine; ∘ means administration of the placebo.

Results

1. Safety Profile of Dosage

Dosage of the composition and the vaccine used in EXAMPLE 5 is based onthe dosage in EXAMPLE 1. Preclinical toxicology studies of the primatesand rodents provide main information for safety profile. EXAMPLE 2evaluates tolerance of the composition, or PIKA only.

Use of commercially available vaccines is also taken into consideration.The commercially available vaccines are generally administrated threetimes in 6 months (10 μg or 20 μg HBsAg). For dialysis subjects andsubjects who are about to receive transplantation, a vaccine isadministrated 4 times (40 μg HBsAg), each of which may be performed inmonth 0, month 1, month 2, and month 6.

Preclinical immunological studies further valid that the recommendeddosages provide adequate immune responses. According to the results,when the ratio of antigen to PIKA equals to or is near 1:25, the humoralimmune responses and T cell mediated immune responses reach adequatebalance. To those skilled in the art, increasing the ratio of antigen toPIKA (1:25) may enhance the humoral immune responses.

To apply a high dosage of HBsAg (40 μg) to the clinical studies, thecomposition includes 1000 μg of PIKA according to the ratio (1:25).According to the results of EXAMPLE 1, the subjects can tolerate 40 μgHBsAg and 1000 μg PIKA. To decrease clinical risks, subjects in thehalf-dosage group are administrated 20 μg HBsAg and 500 μg PIKA. Thesafety profile of the half-dosage group indicates the combination of 40μg HBsAg and 1000 μg PIKA is optimal.

2. Optimized Administration Schedule

By modifying conventional administration schedule (three times in 6months), the composition is more effective. The present disclosurefurther provides an accelerated administration schedule to elicitprotective anti-virus antibody production. More particularly, theaccelerated administration schedule has more frequent of administrationthan the conventional administration schedule, as a result of which, theaccelerated administration schedule may elicit T cell mediated immuneresponses. In EXAMPLE 5, the administration schedule (three times in 2months) provides enough time to decrease risk of subjects between twoadministration intervals, and enough time to observe immune responses.

3. Antibody Titers

Results of the humoral immune responses are based on evaluation of theblood samples of each group collected on day 0, day 56, day 84, and day196. The blood samples are evaluated to acquire titers of the anti-HBVantibodies. The results show immunogenicity of the composition over thecontrol group (group C, commercial available vaccine ENGERIX™-B).According to Table 6, the results indicate both full-dosage group (GroupB) and half-dosage group (Group A), once applied the acceleratedadministrated schedule, generate stronger and earlier immune responses.

TABLE 6 Numbers and percentages of subjects who obtain titer of 10mIU/ml The composition Numbers and percentages of subjects who obtainHBsAg PIKA Alum titer of 10 mIU/ml Group N (μg) (μg) (μg) Day 0 Day 56Day 84 Day 196 A 11 20 500 — 0 (0%) 8 (72.73%) 10 (90.91%) 10 (90.91%) B10 40 1000 — 0 (0%) 10 (100%)   10 (100%)  10 (100%)  C 11 20 — 500 0(0%) 6 (54.55%)  7 (63.64%)  9 (81.82%) N means number of the subjectsin a group

WHO recommends that standard titer of anti-virus antibodies should be 10mIU/ml to provide protection from HBV infection. According to Table 6,on day 56 (i.e., after the second administration), all subjects in thefull-dosage group have obtained the standard titer, while 72.73% of thesubjects in the half-dosage group and 54.55% of the subjects in thecontrol group have obtained the standard titer. On day 196, percentagesof the subjects having the standard titer in full-dosage group,half-dosage group and control group are 100%, 90.91%, and 81.82%,respectively.

TABLE 7 Numbers and percentages of subjects who obtain titer of 150mIU/ml The composition Numbers and percentages of subjects who obtainHBsAg PIKA Alum titer of 150 mIU/ml Group N (μg) (μg) (μg) Day 0 Day 56Day 84 Day 196 A 11 20 500 0 (0%) 3 (27.27%) 9 (81.82%) 10 (90.91%) B 1040 1000 0 (0%) 4 (40%)   10 (100%)   10 (100%)  C 11 20 500 0 (0%) 4(36.36%) 4 (36.36%)  5 (45.45%) N means total number of the subjects

According to Table 7, subjects in full-dosage group (Group B) andhalf-dosage group (Group A) acquire stronger humoral immune responses onday 84 and day 196, respectively. On day 84, all subjects in full-dosagegroup obtain titer of antibodies over 150 mIU/ml; 81.82% of the subjectsin half-dosage group, and 36.36% of the subjects in control group,obtain titer of antibodies over 150 mIU/ml. On day 196, percentages ofsubjects who obtain titer over 150 mIU/ml in full-dosage groups,half-dosage group, and control group are 100%, 90.91%, and 45.45%,respectively.

The clinical studies further investigate HBV specific T cell mediatedimmune responses. The HBV specific T cell mediated immune responses areevaluated by Spot Forming Unit (SFU) in each well (including 10⁵ cells).As illustrated in FIG. 5 , on day 56, average SFU of the subjects infull-dosage group (Group B) and half-dosage group (Group A) are 230 and138, respectively, while no SFU is observed in control group (Group C).On day 84, the average SFU of the subjects in full-dosage group andhalf-dosage group are 134 and 157, respectively, while the average SFUof subjects in control group is 186. On day 196, the average SFU of thesubjects in full-dosage group and half-dosage group are similar to thoseon day 84, while the average SFU of subjects in control group is only24.

Furthermore, cytokine production of CD4⁺ T cells from one subject chosenaccording to results of ELISPOT is evaluated by flow cytometry. Asillustrated in FIG. 7 , under stimulation of one single peptide, CD4⁺ Tcells from subjects in full-dosage group (Group B) express threecytokines (including IFN-γ, TNF-α, and IL-2), while CD4⁺ T cells fromsubjects in half-dosage group (Group B) and control group (Group C)express two of the three cytokines. FIG. 6 illustrates flow cytometryresult of one subject in full-dosage group.

The results of the clinical studies indicate the full-dosage group andthe half-dosage group show adequate tolerance to the composition.High-titer humoral immune responses and T cell mediated immune responsesindicate the composition may elicit effective protection in chronic HBVsubjects.

Example 6: Effects of the Composition on HBV Infected Patients

Patient One:

Mr. Zhao, male, 39 years old, has been infected with HBV (HBsAg+;HBeAg+; HBcAb+) for 9 years. He received anti-virus treatments by takingLamivudine or Entecavir. After stopping taking anti-virus treatments for2 months, a test showed that concentration of HBV-DNA was 3.6×10⁶copies/ml; concentration of HBsAg was greater than 225 ng/ml;concentration of HBeAg was greater than 76.5 PEIU/ml; anti-HBeAgantibody was negative; concentration of anti-HBcAg antibody was 3PEIU/ml. The composition was administrated once every 2 weeks or 4weeks. The composition including 40 μg HBsAg and 1000 μg PIKA wasadministrated each time. From day 112 after first administration to day330 (final administration), dosage of the composition increased to 50 μgHBsAg and 1500 μg PIKA. From day 260, the concentration of the HBV-DNAstarted to decrease. On day 302, the concentration of the HBV-DNA was1.5×10⁴ copies/ml. Meanwhile, the concentration of HBeAg slowlydecreased. After 2 years since first administration, the concentrationof HBeAg reached 38.27 PEIU/ml. During administration of thecomposition, on day 42 concentration of ALT and AST showed a temporaryraise, and from day 260 the concentration was back to normal.

Patient Two:

Mr. Song, male, 46 years old, has been infected with HBV (HBsAg+;HBeAb+; HBcAb+) for 4 years. He received anti-virus treatments by takingAnti-HBV TF. Before administration of the composition, a test showedthat concentration of HBV-DNA was 6.0×10³ copies/ml; concentration ofHBsAg was greater than 225 ng/ml; HBeAg was negative; concentration ofanti-HBeAg antibody was greater than 2.0 PEIU/ml; concentration ofanti-HBcAg antibody was greater than 3.9 PEIU/ml; ALT was abnormal (57U/L). The composition was administrated once every 2 weeks or 4 weeks.The composition including 40 μg HBsAg and 1000 μg PIKA was administratedeach time. Starting from day 112 after first administration, dosage ofthe composition increased to 50 μg HBsAg and 1500 μg PIKA. On day 171,the concentration of the HBV-DNA decreased (<500 copies/nil). Theconcentration of HBsAg went down to 77.35 ng/ml, and ALT was back tonormal. From day 246 to day 330, the composition including 60 μg HBsAgand 2000 μg PIKA was administrated once every two weeks or four weeks.After 2 years since first administration, the concentration of HBV-DNAwas lower than 500 copies/ml, the concentration of HBsAg was 3.17 ng/ml,and the concentration of ALT went back to normal.

Patient Three:

Mr. Zhang, male, 32 years old, has been infected with HBV (HBsAg+;HBeAb+; HBcAb+) for 8 years. Before administration of the composition, atest showed that HBV-DNA was negative; concentration of HBsAg wasgreater than 225 ng/ml; HBeAg was negative; concentration of anti-HBeAgantibody was greater than 2.0 PEIU/ml; concentration of anti-HBcAgantibody was greater than 3.9 PEIU/ml. The composition was administratedonce every 2 weeks or 4 weeks. The composition including 40 μg HBsAg and1000 μg PIKA was administrated each time. Starting from day 112 afterfirst administration, dosage of the composition increased to 50 μg HBsAgand 1500 μg PIKA. On day 171, the concentration of the HBsAg went downto 49.86 ng/ml.

Example 7. Effects of Different Types of the Composition

In EXAMPLE 7, 18 HBV-transgene mice which express high-level HBV DNA areused. All mice are divided into three groups (group A, group B, andgroup C), each of which includes 6 mice. According to Table 8, thecompositions and other substances are administered to each group of themice. The compositions and other substances are administered to the miceonce a week for 12 weeks. A seven-day observation is performed after thefinal administration. The body weight of each mouse is measured twice aweek. After the first administration, the HBV DNA is measured on day 0,day 21, and day 42, respectively.

TABLE 8 The compositions administrated to the mice The compositionsGroup name PBS PIKA HBsAg HBcAg group A + group B 50 μg 10 μg group C 50μg 10 μg 10 μg

According to FIG. 8 , even in the mice expressing high-level HBV-DNA,after the compositions are administrated to the mice from the group Band group C, HBV DNA decreases, which indicates that the compositionssignificantly prevent and treat HBV infection.

It should be noted that the EXAMPLE 7 is merely by way of example. Thoseskilled in the art may change the form and details of the EXAMPLE 7without departing the spirit and scope of the present disclosure. Forexample, ratio of the HBsAg and/or HBcAg to PIC (or PIKA) may be 1:50,1:40, 1:30, 1:25, 1:20, 3:50, 1: 15, 1:10, 1:5, or any value between1:50 and 1:5.

Example 8. Stability of the Composition

In EXAMPLE 8, three batches of the composition are used. Each batch isdivided into two groups (two groups are placed in an incubator adjustedto a temperature of 37° C., and 25° C., respectively). A hepatitis Bvaccine which includes Alum adjuvant and HBsAg is used as control. Thegroup of the composition placed at 37° C. for 1 week, 2 weeks, and 4weeks is serially diluted and administrated to the mice. Blood samplesare collected to measure the HBsAb. Ratio of ED₅₀ of the composition andthe control vaccine is measured. The group of the composition placed at25° C. for 1 month, 2 months, 3 months and 4 months is serially dilutedand administrated to the mice. Blood samples are collected to measurethe HBsAb. Ratio of ED₅₀ of the composition and the control vaccine ismeasured.

According to Table 9, after placed at 37° C., the composition of thepresent disclosure shows higher potency than the control hepatitis Bvaccine; after placed at 25° C., the composition of the presentdisclosure also shows higher potency and stability than the controlhepatitis B vaccine.

TABLE 9 Stability study of the composition Batch Potency Alum + HBsAgPIKA + HBsAg Temperature Time/duration 1 2 3 1 2 3  4° C. Day 0 6.2 6.24.4 8.2 7.4 11.7 37° C. 1 week 4.2 4.5 5.6 5.0 6.7 2.9 2 weeks 4.9 3.84.1 10.1 10.1 4.4 4 weeks 3.9 2.4 3.2 4.5 4.0 7.9 25° C. 1 month 6.3 5.65.4 10.9 24.2 7.9 2 months 5.4 5.4 5.7 13.1 9 7.4 3 months 3.0 4.4 3.914.1 17.9 7.1 6 months 1.7 2.3 1.8 18.3 9.3 9.9

It should be noted that although one type (i.e., composition includingPIKA and HBsAg) of the composition is used in Example 8, the EXAMPLE 8is illustrative and that the scope is not limited. To those skilled inthe art, various changes in form and details of the EXAMPLE 8 may bemade without departing from the scope of the present disclosure. Merelyby way of example, another type (e.g., composition including PIKA, HBsAgand HBcAg) of the composition may be used in the EXAMPLE 8. For anotherexample, those skilled in the art may use a different concentration ofthe composition, or a different concentration of each substance of thecomposition. For example, ratio of the HBsAg and/or HBcAg to PIC (orPIKA) may be 1:50, 1:40, 1:30, 1:25, 1:20, 3:50, 1: 15, 1:10, 1:5, orany value between 1:50 and 1:5.

According to the present disclosure, the composition provides at leastone of following advantages:

-   -   good safety both in acute and chronic toxicological studies;    -   enhancing humoral immune responses, and generating higher titer        and earlier antibody production;    -   effectively triggering T cell mediated immune responses;    -   decreasing HBV DNA load in HBV infected subjects.

While the present disclosure has been described with reference toparticular embodiments, it will be understood that the embodiments areillustrative and that the scope is not limited. Alternative embodimentsof the present disclosure will become apparent to those having ordinaryskill in the art to which the present disclosure pertains. Suchalternate embodiments are considered to be encompassed within the spiritand scope of the present disclosure.

What is claimed is:
 1. A composition comprising: polyriboinosinicacid-polyribocytidylic acid (PIC), at least one antibiotic or polyamidecompound, at least one positive ion, and HBsAg, wherein theconcentration of the HBsAg in the composition ranges from 20 μg/unitdose to 60 μg/unit dose.
 2. The composition of claim 1, furthercomprising HBcAg.
 3. The composition of claim 1, wherein the HBsAg isproduced by Hansenula polymorpha, CHO cells, insect expression system,Saccharomyces cerevisiae, or Pichia pastoris.
 4. The composition ofclaim 2, wherein the HBcAg is produced by Escherichia coli, Hansenulapolymorpha, insect expression system, Saccharomyces cerevisiae, orPichia pastoris.
 5. The composition of claim 1, wherein the antibioticis selected from a group including tobramycin, anthracycline, butyrosinsulfate, gentamicin, hygromycin, amikacin, dibekacin, nebramycin,beta-lactam, neomycin, puromycin, streptomycin, streptozotocin, andkanamycin.
 6. The composition of claim 1, wherein the polyamide compoundis selected from a group including spermidine salt, spermidine,N-(3-aminopropyl), N-(3-aminopropyl)-1,4-butandiamine, spermine BR,spermine, OS-dimethylphosphoramidothioate, polylysine, andaminoglycoside.
 7. The composition of claim 1, wherein the positive ionis selected from a group including calcium, cadmium, lithium, magnesium,cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, andzinc.
 8. The composition of claim 1, wherein ratio of the HBsAg to PICis 1:30, 1:25, or 1:20.
 9. The composition of claim 2, wherein ratio ofthe HBcAg to PIC is 1:30, 1:25, or 1:20.
 10. The composition of claim 1,wherein concentration of PIC in the composition ranges from 250 μg/unitdose to 6000 μg/unit dose.
 11. The composition of claim 1, whereinconcentration of the PIC in the composition ranges from 500 μg/unit doseto 1500 μg/unit dose.
 12. The composition of claim 2, whereinconcentration of the HBcAg in the composition ranges from 10 μg/unitdose to 100 μg/unit dose.
 13. The composition of claim 10, wherein oneunit dose ranges from 0.1 ml to 250.0 ml.
 14. The composition of claim1, wherein the composition further comprises at least one substanceselected from a group including gelatin, saccharose, granulated sugar,lactose, maltose, trehalose, glucose, low molecular dextran, sorbitol,polysorbate 20, mannitol PEG, human blood albumin, recombinant humanserum albumin, sodium caprylate, urea, aluminum hydroxide, phenol red,magnesium chloride, potassium chloride, sodium chloride, sodiumthiosulphate, potassium dihydrogen, ascorbic acid, trichloromethane,phenol, and thiomersal or at least one physiologically acceptablebuffer, wherein the physiological acceptable buffer is selected from agroup including acetate buffer, trisamine buffer, bicarbonate buffer,carbonate buffer, and phosphate buffered saline.
 15. The composition ofclaim 1, wherein pH of said composition ranges from 6.0 to 8.0.
 16. Thecomposition of claim 1, wherein the composition is in a liquid or solidform, wherein said liquid form is selected from a group includingparenteral solution, suspension, ointment, emulsion, drop, syrup, andgel; wherein said solid form is selected from a group including drypowder, freeze-dried powder, tablet, capsule, suppository, granule, andsugar-coated tablet.
 17. A method for treating and/or preventionHepatitis B virus infection, comprising administering a composition to asubject, wherein said composition comprises PIC, at least one antibioticor polyamide compound, at least one positive ion, and HBsAg, wherein theconcentration of the HBsAg in the composition ranges from 20 μg/unitdose to 60 μg/unit dose.
 18. The method of claim 17, said method furthercomprising administering the composition to the subject once every twomonths, twice every two months, three times every two months, four timesevery two months, five times every two months, once every month, twiceevery month, once every three weeks, twice every three weeks, twiceevery two weeks, once every two weeks, or once every week or wherein thecomposition is administered to the subject by one way selected from agroup including intramuscular injection, intraperitoneal injection,intravenous injection, subcutaneous injection, transdermal delivery,intradermal delivery, nasal delivery, opthamalical delivery, oraldelivery, sublingual delivery, peritumoral delivery, and intratumordelivery.
 19. The method of claim 17, wherein said subject is a HBVuninfected subject, a chronic HBV subject, or an acute HBV subject. 20.The method of claim 17, wherein the concentration of the HBsAg in thecomposition ranges from 40 μg/unit dose to 60 μg/unit dose and theconcentration of the PIC in the composition ranges from 1000 μg/unitdose to 1500 μg/unit dose.
 21. The composition of claim 1, wherein thecomposition comprises an antibiotic, wherein the antibiotic compriseskanamycin and the positive ion comprises calcium.